The present invention relates to a method of testing the toxicity of a chemical substance in particular with reference to animal, especially human mucosal membranes, more especially single-layered mucosal surfaces such as human intestinal, nasal and respiratory mucosa and other mucosal surfaces such as vaginal and buccal mucosa.
Higher animals, such as mice, rats, rabbits, dogs, cats, pigs, and monkeys, are usually used as test animals in conducting toxicity tests of substances. Breeding must be carried out under carefully controlled conditions over a long period of 2 to 2xc2xd years. Thus, performing such toxicity tests is very time consuming and expensive. In the chemical and pharmaceutical industries, novel chemical substances are continually being synthesised to meet various industrial and health needs. Additionally, much research is being done to find new applications for known chemical substances. In each case, it is desirable to establish a rapid and inexpensive method for testing toxicity of these substances.
Toxicity testing using vertebrate animals to evaluate the safety of xenobiotics to humans has been severely criticised based on ethical and financial considerations. The principle alternative to in vivo animal testing is in vitro testing. Many factors such as nervous control, systemic blood flow, reduced motility and heterogeneous cell populations, however, are absent in simple cell culture models which reduces the value of in vitro testing. Bacteria, for instance, are prokaryotic organisms which are quite different from multicellular organisms which possess higher levels of biological organisation. There is therefore a tendency to use higher animals or parts of higher animals for testing of complex systems such as human mucosa.
In the workshop xe2x80x9cThe three Rs: The Way Forwardxe2x80x9d organised by the European Centre for the Validation of Alternative Methods (ECVAM) (Sheringham, Norfolk, UK, 30/5-3/6, 1995) some replacement alternative methods and approaches were proposed, for example, the use of xe2x80x9clowerxe2x80x9d organisms with limited sentience and/or not protected by legislation controlling animal experiments, including invertebrates, plants and micro-organisms. However, only nematodes, bacteria or insects were proposed as concrete alternatives to animal testing. The use of nematodes for toxicity testing is described in U.S. Pat. No. 4,444,891.
Investigations have demonstrated that the human mucosa, in particular the nasal mucosa allows effective drug absorption. Particularly for peptide drugs, nasal delivery is a promising alternative to parenteral administration. However, because of their high molecular size and hydrophilic properties at physiological pH values, peptides show poor transport characteristics across the hydrophobic membrane barriers. The absorption efficiency of intranasally administered peptides can be improved by the use of absorption enhancers, such as bile salts, laureth-9, fusidate derivatives or sodium taurodihydrofusidate (STDHF).
Such absorption enhancers play an essential role in nasal drug delivery. In particular, for peptides and proteins the use of absorption enhancers is often mandatory to reach an effective absorption from the nasal mucosa. Unfortunately, little is known about potential side effects of these substances, which is a drawback for the clinical application of absorption enhancers in nasal drug formulations. Looking more closely at the effects and the mechanisms underlying absorption enhancement of absorption enhancers on the nasal mucosa provides more information on their efficacy and safety. The mechanisms that lead to increased nasal drug absorption under the influence of enhancers are quite diverse and only partly understood. In some cases the solubility or the stability of the drug is increased, but absorption enhancers can also interact with the mucus layer, changing the mucus properties. Furthermore, the permeability of the nasal epithelium might also be increased due to interaction with the epithelial membranes.
When considering nasal drug delivery, the effects of drug and additives on nasal functions should be tested at an early stage. Because the self-cleaning capacity of the nose by the ciliary epithelium is vital for the removal of dust, allergens and bacteria from the nasal area of humans, it should not be influenced by nasal medication. Ciliary movement is a major factor for the mucociliary clearance in the upper airways. From patients with xe2x80x9cimmotile cilia syndromexe2x80x9d it is known that chronic nasal ciliary arrest leads to recurrent infections of the airways. Many drugs and additives inhibit nasal ciliary movement as demonstrated in vitro. For instance, lipophilic and mercuric preservatives, antihistamines, propranolol and dihydroxy bile salts are ciliostatic agents, all reducing the ciliary beat frequency within a short time. It is further important to investigate whether a ciliostatic effect is reversible after withdrawal of drug exposure. Nasal absorption enhancers should be devoid of any serious ciliotoxicity, and the feasibility of nasal drug administration may depend largely on the effects on the ciliated epithelial tissue.
The following methods for determining the cytotoxicity or mucociliary clearance time of chemical substances have been proposed: in situ tests on the rat nasal epithelium, in vitro determination of the influence of enhancers on the ciliary beat frequency of cilia of new-born chicken trachea and the use of the frog palate model in which the frog is decapitated, the upper palate is exposed and tested visually with the specific substance.
Not only nasal functions but also negative side effects should be determined at an early stage in drug or drug delivery development, ideally before clinical trials on humans. An example is the delivery of the xcex2-blocker propranolol HCl via human buccal mucosa. This chemical caused severe ulceration of humans during clinical trials that took weeks to heal. It would be desirable to have a screening test method which could reliably screen out chemicals which cause such serious problems to humans.
Gastropods are molluscs and include snails and slugs. They generally have a single shell or none at all and often have an asymmetric body. A more extensive discussion of gastropods may be found in the Encyclopaedia Britannica. Until now gastropods, e.g. snails, have mainly be used in medical products and processes as described, for instance in U.S. Pat. Nos. 5,538,740, 4,473,640, 4,314,992, 3,889,006, 4,855,285, 3,885,012.
In the article by Marigomez et. al., Arch. Environ. Contam. Toxicol. 1996, 31(1), pages 54-62, the use of slugs as sentinel organisms (xe2x80x9cSlug Watchxe2x80x9d) is described in soil quality assessment. The slugs may be used as a biomarker of exposure to metallic pollutants in the soil. Reference is only made to the reaction of slugs to mercury and there is no indication that the slugs may be used as a model for human mucosa.
It is an object of the present invention to provide a test method with which substances may be tested for their toxicity to animal and human membranes easily, rapidly, accurately and inexpensively.
An objective of the present invention is to provide a method for testing the toxicity of a substance using invertebrates in lieu of vertebrates.
Another objective of the present invention is to provide a test allowing easy replication of tests to improve the statistical interpretation of the results.
The present invention includes use of a gastropod for toxicity testing. The present invention includes a toxicity test method for modelling the irritation to vertebrate mucosal surfaces, in particular to human or other mammal mucosal surfaces, by a chemical substance comprising the steps of:
providing at least a part of the epithelium of the body or a part of the foot of a gastropod, a gastropod being an organism of the class gastropoda; and
exposing the part of the epithelium or the part of the foot to the chemical substance to be tested.
In particular, the chemical substance may be brought into contact with the skin and/or the foot of the gastropod. The present invention includes both in vivo tests using the whole gastropod or in vitro tests on a part of a gastropod. The chemical substances are not limited to cosmetic or pharmaceutical chemical formulations but may be household or industrial items such as paints, lacquers, adhesives and solvents which may come into contact with the human or animal mucosal membranes.
According to this test the mucosal irritation can be easily and rapidly determined qualitatively, e.g. by visual observation of the colour or increase in the amount of mucus liberated from the foot of the gastropod. Mechanical vibration and other irritations may affect mucus production of gastropods. To avoid confusion with other effects it is preferred in accordance with the present invention if the gastropods are not subjected to other forms of stress or agitation during the tests except for the chemical substances to be tested. According to the toxicity test method of the present invention, mucosal damage can also be easily determined quantitatively by the release of mucus from the foot, by the loss of body weight of the gastropod, or by release of marker substances, proteins or enzymes from the foot. In particular, the toxicity of a substance can be determined quantitatively by measuring the amount of total protein and/or lactate dehydrogenase and/or alkaline phosphatase and/or cholesterol and/or phospholipids and/or other membrane components released from the foot mucosa of the gastropod after treatment with the chemical substance. An advantage of the test is that the mucus production caused by irritating substances can be simply measured. The method in accordance with the present invention provides a simple screening test for the toxicity of substances while also providing a model for single-layered mucosal surfaces such human intestinal, nasal and respiratory mucosa and other mucosal surfaces such as vaginal and buccal mucosa, which can allow accurate quantitative analysis. Hence, the test method of the present invention may provide both qualitative and quantitative test results, e.g. the release of proteins, enzymes or membrane components from the treated epithelium, and may be suitable as both a screening test and an accurate quantitative toxicity test.
The present invention will be described with reference to the following figures. The dependent claims define individually, separate embodiments of the present invention.